Diabetic cardiomyopathy is characterized by profound alterations in cardiac lipid metabolism resulting in deleterious changes in the chemical composition and biophysical properties of cardiac membranes. These covalent membrane modifications lead to maladaptive changes in cellular signaling cascades, compromise cardiac myocyte bioenergetic efficiency, and alter the kinetics of critical transmembrane proteins that orchestrate cardiac electrophysiologic properties, excitation-contraction coupling, and diastolic function. In the diabetic state, the excessive reliance on fatty acids as substrate to fuel contractile function leads to the persistent and ultimately maladaptive activation of a family of calcium-independent phospholipases (iPLA2s) that hydrolyze membrane lipids, detrimentally modify membrane lipid composition, and precipitate membrane dysfunction. In previous work, we have identified the three major iPLA2 activities in myocardium, including iPLA2?, iPLA2??and iPLA2??and have implicated these proteins as the enzymic mediators of membrane dysfunction in diabetic myocardium through multiple chemical, biologic, pharmacologic and genetic approaches. In the proposed studies, we will dissect the role of each of these enzymes in the pathologic sequelae of diabetic cardiomyopathy. First, we will determine the detailed molecular mechanisms through which acyl-CoA activates iPLA2? in diabetic and/or ischemic myocardium through exploiting transgenic mice overexpressing iPLA2?. The causal role linking the observed covalent changes in iPLA2? to diabetic myocardial dysfunction and the lethal sequelae of myocardial ischemia will be determined through targeted site-directed mutagenesis experiments where we will ablate activation of iPLA2? in diabetic and ischemic myocardium. In Specific Aim 2, we will examine the hypothesis that specific isoforms of iPLA2? mediate interactions between peroxisomal and mitochondrial energy production, energy storage, lipid synthesis and lipid removal (by heat generation) that coordinately regulate cardiac metabolic flexibility during health, but are compromised in the diabetic heart. The roles of iPLA2? as a signaling enzyme communicating mitochondrial functional status to other cellular compartments will be examined by organelle-specific genetic rescue. The biochemical mechanisms that alter membrane function resulting from changes in iPLA2? activity leading to intolerance to metabolic stress will be identified and causality determined by rescue experiments. In Specific Aim 3, the roles of iPLA2 family members in mediating alterations in triglyceride metabolism, metabolic flux and alterations in triglyceride recycling in the diabetic state will be explored using stable isotope experiments in conjunction with high resolution mass spectrometry. The hypothesis that iPLA2? catalyzes triglyceride hydrolysis in ischemic myocardium will be tested using iPLA2? -/- mice. Through the synergistic use of genetically-engineered mice with informative physiologic models analyzed by shotgun lipidomics and stable isotope methods, changes in lipid metabolism mediated by iPLA2 family members leading to membrane dysfunction which precipitates diabetic cardiomyopathy will be defined on a chemical level identifying novel PLA2 exosites as new pharmaceutical targets. PUBLIC HEALTH RELEVANCE Diabetic heart disease is growing in endemic proportions in industrialized societies. During diabetes, the heart undergoes profound changes in metabolism that lead to alterations in the regulation of lipid metabolism and cell signaling. This proposal examines the pathologic interplay between altered energy metabolism and membrane dysfunction in diabetic hearts. More specifically, the proposed studies identify the chemical changes in hearts of diabetic patients that lead to altered heart function and ultimately to heart failure. [unreadable] [unreadable] [unreadable]